Dual effect of tamoxifen, an anti-breast-cancer drug, on intracellular Ca²⁺ and cytotoxicity in intact cells

The effect of tamoxifen on Ca²⁺ signaling and viability in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca²⁺ probe. Tamoxifen evoked a rise in cytosolic free Ca²⁺ levels ([Ca²⁺](i)) concentration-dependently between 1 and 50 μM with an ECS0 of 10 μM. The response was decreased by extracellular Ca²⁺ removal. In Ca²⁺-free medium, pretreatment with 5 μM tamoxifen abolished the [Ca²⁺](i) increase induced by the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin (1 μM), but pretreatment with brefeldin A (50 μM; a Ca²⁺ mobilizer of the Golgi complex), thapsigargin (an inhibitor of the endoplasmic reticulum Ca²⁺ pump), and carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler), only partly inhibited tamoxifen-induced [Ca²⁺](i) increases. This suggests that tamoxifen released Ca²⁺ from multiple pools. Addition of 3 mM Ca²⁺ induced a [Ca²⁺](i) rise after pretreatment with 5 μM tamoxifen in Ca²⁺-free medium. Inhibiting inositol 1,4,5-trisphosphate formation with the phospholipase C inhibitor U73122 (2 μM) did not alter 5 μM tamoxifen-induced Ca²⁺ release. The [Ca²⁺](i) increase induced by 5 μM tamoxifen was not altered by La³⁺, nifedipine, verapamil, or diltiazem. Tamoxifen (1-10 μM) decreased cell viability in a concentration- and time-dependent manner. Tamoxifen (5 μM) also increased [Ca²⁺](i) in neutrophils, bladder cancer cells, and prostate cancer cells from humans and glioma cells from rats. Collectively, it was found that tamoxifen increased [Ca²⁺](i) in MDCK cells by releasing Ca²⁺ from multiple Ca²⁺ stores in a manner independent of the production of inositol 1,4,5-trisphosphate and also by triggering Ca²⁺ influx from extracellular space. The [Ca²⁺](i) increase was accompanied by cytotoxicity. (C) 2000 Academic Press.